Binary Defense 2017 p. 1 Binary Defense: Cyanogenic Glycosides and Glucosinolates (toxic products in common food plants) Background: recall plant phytochemical defensive strategies encountered i) constitutive defenses (always present many alkaloids and triterpenes) ii) induced defenses (i.e, nicotine, phytoalexins, protease inhibitors) iii) "binary" defense (preformed but not active) today's lecture I. Cyanogenic Glycosides 1. Definition: Glycosides which release HCN when sugar is removed widespread in plants, but in particular in Rosaceae (almonds, peach seed), Fabaceae (Lotus, clover), Graminae (sorghum) found in at least 2000 plant species, 60 different structures have been described. evolved independently in different taxa. 2. Typical Structure: three components: nitrile, glucose (or other sugar), amino acidderived Rgroup (variable) amino acid derived, often the Rgroup is still very similar 3. Simple Biosynthesis: derived from one of five amino acids: aromatic (Phe, Tyr) or branchedchain (Leu, Ile, Val) steps: Nhydroxylation/decarboxylation (P450, aldoxime) (multifunctional P450) nitrile formation (P450) (multifunctional P450) glucosylation by UDPdependent glycosyltransferases (the presence of the glucose stabilizes nitrile) 4. Mechanism of cyanogenesis (HCN production): both spontaneous and enzymemediated enzyme kept in separate compartment from the compound itself (vacuolar) i) enzyme removes glucose: ßglucosidase (can also happen spontaneously) ii) 2hydroxynitrile product is unstable > hydroxynitrillyase (optional) > HCN is released Compartmentalization of cyanogenic glycoside and the respective ßglucosidase is essential! (=binary defense)
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Binary Defense 2017 p. 1
Binary Defense: Cyanogenic Glycosides and Glucosinolates (toxic products in common food plants)
Background: recall plant phytochemical defensive strategies encountered i) constitutive defenses (always present -‐ many alkaloids and triterpenes) ii) induced defenses (i.e, nicotine, phytoalexins, protease inhibitors) iii) "binary" defense (preformed but not active) -‐ today's lecture
I. Cyanogenic Glycosides 1. Definition: Glycosides which release HCN when sugar is removed -‐ widespread in plants, but in particular in Rosaceae (almonds, peach seed), Fabaceae (Lotus, clover), Graminae
(sorghum) -‐ found in at least 2000 plant species, 60 different structures have been described. -‐ evolved independently in different taxa. 2. Typical Structure: -‐ three components: nitrile, glucose (or other sugar), amino acid-‐derived R-‐group (variable) -‐ amino acid derived, often the R-‐group is still very similar
3. Simple Biosynthesis:
-‐ derived from one of five amino acids: aromatic (Phe, Tyr) or branched-‐chain (Leu, Ile, Val) -‐ steps: -‐ N-‐hydroxylation/decarboxylation (P450, aldoxime) (multifunctional P450) -‐ nitrile formation (P450) (multifunctional P450) -‐ glucosylation by UDP-‐dependent glycosyltransferases (the presence of the glucose stabilizes nitrile)
4. Mechanism of cyanogenesis (HCN production): -‐ both spontaneous and enzyme-‐mediated -‐ enzyme kept in separate compartment from the compound itself (vacuolar) i) enzyme removes glucose: ß-‐glucosidase (can also happen spontaneously) ii) 2-‐hydroxynitrile product is unstable -‐> hydroxynitrillyase (optional) -‐-‐> HCN is released Compartmentalization of cyanogenic glycoside and the respective ß-‐glucosidase is essential! (=binary defense)
20. Binary Defenses p. 2
6. Defensive Functions and Counteradaptations (Examples) NB: HCN is a general inhibitor of mitochondrial respiration i. Clover (Lotus) contains linamarin -‐ genetic polymorphisms: low and high HCN genotypes -‐ higher slug herbivore pressure at lower latitudes favor high HCN genotypes (early season herbivory) ii. Cassava (Manioc esculenta) contains dhurrin -‐ cassava root is a tropical starchy staple (we used it mostly for its starch in tapioca) -‐ root crop, perennial, good in poor soils, pest-‐resistant -‐ bitter and sweet varieties relate to dhurrin content -‐ processing roots by grating and soaking followed by drying or heating detoxifies (removes HCN) -‐ humans have rhodanese enzyme to detoxify HCN (induced) (NB: lethal dose of dhurrin: 0.5-‐3.5 mg/kg body wt) iii. Almond, apple, peach (Rosaceae) seeds contain amygdalin & prunasin) -‐ bitter almonds are toxic but used for flavor (benzaldehyde) -‐ 'sweet' almonds have mutation and less amygdalin -‐ cyanogenic prune or peach stones: ~300 mg HCN /100 g -‐ apple seeds: ~60mg/100g ( 2 cups = lethal dose??) iv. Insect Counteradaptations -‐ sequestration and storage linamarin from Lotus (clover) by burnet moth larvae. These insects have
additionally independently evolved their own linamarin synthesis pathway (convergent evolution). -‐ Heliconius larvae detoxify cyanogenic glycosides (see below)
7. Additional functions in N transport -‐ Attempts to reduce linamarin in transgenic cassava led to the realization that these compounds are
transported, and used by the plant as transport forms.
20. Binary Defenses p. 3 -‐ in this case they function as primary as well as secondary metabolites. II. Glucosinolates (Thioglucosides, or Mustard Oils) (= sulfur containing glycosides) 1. Introduction
-‐ amino acid derived (aromatic, aliphatic) -‐ limited distribution (Brassicaceae: Brassica, Arabidopsis, etc) -‐ they are very important in the human diet (flavor, health) -‐ interact with enzyme = binary defense) à lead to isothiocyanate and related compounds
2. Structure Three components: thioglucoside, hydroximinosulfate ester, & variable R-‐group (amino acid-‐derived) -‐ based on Phe & Tyr (aromatic), Trp (indole), Leu, Ile, Val (aliphatic ) -‐ 120 structures known (evolved from cyanogenic glycosides?)
3. Biosynthesis -‐ amino acid: decarboxylation/N-‐hydroxylation -‐ add S from cysteine -‐ glucosylation (UDP-‐glucose) -‐ add SO4
-‐2 from PAPS
(PAPS = phosphoadenosine phosphosulfate = key sulfur donor) 4. Hydrolysis products and biological effects -‐ hydrolysis by the enzyme myrosinase (= thioglucosidase) removes glucose. -‐ the aglycone part of the molecule is unstable à leads to spontaneous rearrangements -‐ isothiocyanate is main product, but smaller amounts of other products are formed. These include
thiocyanate, nitriles, epithionitriles -‐ the products that are ultimately produced depend on the R-‐groups, and the presence of additional proteins:
20. Binary Defenses p. 4 i) epithiospecifier protein (ESP) directs rearrangements to epithionitrile ii) works with ESM1 (epithiospecific modifier protein) 5. Biological Effects of glucosinolates, and breakdown products. -‐ products are lipophilic -‐> they penetrate membranes, irritate nerves, impact ion channels ... 'wasabe' -‐ isothiocyanates attack -‐SH, -‐NH3 groups in biomolecules -‐ compounds act as general antifeedants and toxins -‐ good examples of specialist insects that have learned to adapt. i) Isothiocyanates are toxic to generalist pests -‐ toxic effects of sinigrin to swallowtail larvae shown via clever "celery infiltration" experiment: 0.1% sinigrin
=100% mortality ii) Some well-‐studied specialists are adapted to glucosinolates -‐ cabbage butterfly can only feed and reproduce on Brassicas sinigrin = feeding stimulant for larvae, oviposition stimulant for adults -‐ flea beetles: feeding stimulant is a mixture of chemicals including glucosinolates iii) How specialist insects detoxify glucosinolates -‐ cleave sulfate: prevents myrosinase action (diamondback moth) -‐ redirect ITC products via Nitrile Specifier Protein (NSP) = novel insect protein, in cabbage butterfly -‐ also: examples of sequestration (w. insect-‐derived myrosinase) iii) The importance of glucosinolates in the human diet
-‐ Brassica oleracea (cabbage, cauliflower...) & B. rapa (turnip, bok choy) are common vegetables -‐ B. nigra, Sinapis alba species, (black and white mustards) are condiments (mustard taste due to
isothiocyanate) -‐ breeding led to low glucosinolate content in seed for improved canola oil (also low erucic acid & low sinapine) -‐ anticancer effects ascribed to sulforaphanes (high in brussel sprouts) Sulfurophanes: specialized products of some rearranged glucosinolates linked to anti-‐cancer effects via their
ability to induce phase II enzymes (conjugation of xenobiotics, cell cycle control)